Studies with the protein (actin) which binds ethanol-insoluble metabolic ADP has lead to the discovery of the presence of GDP in the ethanol-insoluble fraction. This GDP can be labeled using 14C-guanine or 32P-orthophosphate in intact platelets. This may be bound to platelet tubulin. In contrast to adenylate metabolism, the metabolism of the guanylate system has not been carefully studied in platelets. We propose to carry out a detailed study of uptake of 3H-guanine and 3H guanosine in human platelets. Km and Vmax for the uptake processes will be determined, the specificity of the systems will be delineated and in particular for guanine, the dependence of the intracellular level of phosphoribosylpyrophosphate will be determined. The ability of platelets to incorporate guanine into guanine metabolites known to be present in human tissues will be studied. Specific enzymes catalyzing the various conversions will be partially characterized. When this basic information in resting cells has been obtained, the behavior of the guanine nucleotides during thrombin-induced secretion will be obtained. Especially the possible consumption of GTP during this process, and the secretion and subcellular localization of metabolically inert GTP will be studied. Similarly the behavior of the guanylate system during metabolic shock will be determined. We will follow up the discovery of ethanol-insoluble GDP by attempting to identify the binding protein and study its properties in greater detail. The kinetics of guanine incorporation into ethanol-insoluble proteins of intact resting or stimulated platelets will be determined.